1. Field
This disclosure is concerned generally with an assay for antibodies to streptococcal organisms and specifically with a novel method of determining the titer of group C streptococcal antibodies in a blood serum sample.
2. Prior Art
Organisms of the genus Streptococcus include a variety of nonmotile, chiefly parasitic, gram-positive bacteria that divide only in one plane, occur in pairs or chains but not packets, and include important pathogens of man and domestic animals. The streptococcal genus has been divided into eight distinct groups labelled A, B, C, D, F, G, H, and K. This disclosure is generally concerned with group C streptococcal organisms.
A very important group C organism, Streptococcus equi, is the causitive agent of a severe respiratory disease of horses referred to as "Strangles". The disease is endemic in most parts of the world and epidemic in the United States. Race and show horses are particularly susceptible to repeated infections due to the stress of travel and exposure to new contacts. The disease begins with a mucopurulent nasal discharge, temperatures of 103.degree.-106.degree. F. and severe inflammation of the upper respiratory mucosa. It finally progresses to lymphadenitis and abscess formation which is sometimes severe enough to restrict air intake and cause suffocation of the animal. Strangles results in extensive loss of condition (loss of weight) as it often runs a course of 4-6 weeks. It is thought that there is a single strain of Streptococcus equi (a Lancefield Type C Strep.) which is responsible for this disease world-wide. See, for example, Bergy's Manual of Determinitive Bacteriology (8th Edition), p. 498 (1974). The only other known susceptible animal is the mouse.
Because of the debilitating and in some cases lethal effects of streptococcal infections in man and other animals, attempts have been made over the years to prepare streptococcal bacterins or bacterin-like preparations which could be used for vaccination purposes. Unfortunately, streptococcal strains tend to be very reactive and it has been noted that certain human streptococcal vaccines (in Groups A and B) have stimulated heart muscle reactions while Group C Strep. equi preparations, in live or inactivated forms, have been noted for their affinity for dermal tissue, producing severe swelling at the injection site. This known reactivity has tended to discourage the commercial use and development of immunizing streptococcal products for man and other animals.
In efforts to produce efficacious vaccines from group C streptococci in general, and with Streptococcus equi in particular, it is important to have available a serological assay to measure the titer of serum antibodies to the group C streptococcal organisms. Unfortunately, no such assay has been available.
It is known that human streptococcal literature describe a bactericidal plate count test to measure human streptococcal antibody levels. See for example, Bisno, Alan L., Infect. Immun. 26, 1172-1176 (1979) and Peterson, Phillip K. et al, J. Infect. Dis. 139, 575-585 (1979). That test relies on complement found in fresh human blood. A similar test has been used by J. B. Woolcock (Infection and Immunity, July 1974, p. 116-122) in determining presence or absence of antibody in rabbits and horses after vaccination with Strep. equi. However, the above test (including numerous modifications) was unable to provide a reproducible quantitative measurement of Strep. equi antibody in our experiments. Other serological assays have been described which include a long chain test (see Woolcock et al cited above) and various immunodiffusion tests (Fischetti, V. A. et al, The Journal of Experimental Medicine, Vol. 144, 1976 p. 32-51). Once again, attempts to develop these tests into quantitative serological assays related to protective antibody were unreliable. The above difficulties posed a major problem in the attempt to develop a safe, effective and non-reactive Strep. equi preparation that could be used to immunize horses since, without an accurate serological assay, there was no way to detect susceptible animals.
Quite surprisingly, we were able to show that a relatively simple passive protection test could be used to actually quantitate serological antibody in a susceptible animal such as the mouse. Details of our test are given below.